1. Field of the Invention
The present invention relates generally to the analysis of molecules. In particular, the invention is a novel immobilization technique and substrate wherein a normally electrically-neutral macromolecule is subjected to a chromatographic or electrophoretic separation in the form of a charged conjugate, followed by covalent attachment to a surface. The retained molecule may then be tested utilizing a variety of probing strategies employing specific bioaffinity molecules. More particularly, the invention relates to covalent immobilization of charge-conjugated carbohydrate molecules to a blotting membrane and subsequent determination of activity or structure.
2. Description of the Background Art
Immobilization of macromolecules on a solid-phase support matrix is a technique which has seen widespread use in affinity applications. Most affinity applications require that the molecule or ligand of interest react with the solid phase surface covalently. In most cases the solid phase surfaces are in the form of chromatographic supports, i.e., beads or particles. Affinity Chromatography. A Practical Approach, P. D. G. Dean, ed, IRL press. Porous membrane substrates have been reported, i.e., diazo activated cellulose (Alwine, J. C., Kemp, D. J. and Stark, G. R., Proc. Natl. Acad SIC U.S.A. 74:5350-5354 (1977)), Immobilon-AV.TM. (Millipore Corp., Bedford, Mass.) (Blankstein, L. A. et al., Am. Clin. Prod. Rev. 4:33-34 (1985), and activated nylon (Huse, K. et al., J. Chromatography 502:171-177 (1990). Non-covalent binding on nitrocellulose, or via ionic (nylon) or hydrophobic polyvinyldifiuoride (PVDF) -based membrane mechanisms are reversible and may not retain the molecule or ligand of interest for the intended application. This can be a problem with small molecules such as peptides, oligonucleotides or oligosaccharides, or where competing ions may wash away the adsorbed ligand.
Covalent immobilization on several blotting surfaces is known. Examples include activated paper (TransBind.TM., Schleicher & Schuell Ltd., Keene, N.H. ) carbodimidazole-actived hydrogel-coated PVDF membrane (Immobilon-IAV.TM., Millipore Corp., Bedford, Mass.), activated nylon (BioDyne.TM., Pall Corp., (Glen Cove, N.Y.), DVS- and cyanogen bromide-activated nitrocellulose. Such surfaces are activated prior to transfer of the molecules from the electrophoresis gel and are reactive only if the molecule is well retained by the surface and sufficient "residence time" on the surface allows for reaction before the surface chemistry decays by hydrolysis or non-specific reaction with components of the electrophoresis system. While adsorbed in the dry state and kept in the dark, these blots are stable. Most applications of these blots, however, require further manipulation, often in aqueous buffer systems. These environments can lead to desorption of the blotted pattern, and thus information and material become lost. UV cross-linking of DNA (Church et al., PNAS 81:1991-1995(1984)) and RNA (Khandjian, et al., Anal. Biochem. 159:227-232 (1986) to nylon membranes is well known, and is thought to proceed via a thymidine radical initiated attack upon membrane primary amines.
U.S. Pat. No. 4,512,896 (Gershoni), Transfer of Macromolecules from a Chromatographic Substrate to an Immobilizing Matrix, discloses a charge-modified hydrophilic porous membrane used to immobilize blotted macromolecules. The preferred membrane material is nylon, and the surface is modified with Hercules R-4308 or Polycup 172, 2002, or 1884. The epoxide group is used to link the surface-modifying agent to the polymer surface, the usual method of binding the polymer coating to the membrane surface. No examples of permanent or covalent attachment of macromolecules are disclosed.
U.S. Pat. No. 5,004,543 (Pluskal et al.) discloses a cross-linked cationic charge-modifying polymer coating on a hydrophobic microporous polymer membrane substrate. The coating has fixed formal positive charge groups and halohydrin groups. The coated membrane is produced by contacting the membrane with an aqueous alkaline organic solution of the polymer Hercules R-4308. Again, no examples of macromolecule blotting are shown.
The blotting of modified carbohydrates to membranes is an area of active interest, as shown by the following discussion. A molecule or ligand successfully retained on a solid phase surface is available to form biospecific or affinity interaction with other macromolecules for the purposes of, for example, purification, chemical modification, or confirmation of biological activity. In the latter case the retained molecule or ligand is then subjected to testing techniques with various labeled protein compounds. Antibodies may be used to identify specific proteins. In the case of retained oligosaccharides or glycans released from a proteoglycan or other source molecule, lectins may be used to uniquely identify the adsorbed carbohydrate.
Determination of the sequence and structure of carbohydrates, specifically oligosaccharides, can be of significant importance in various fields, particularly in the medical and pharmaceutical fields. For example, the carbohydrate structure of a glycoprotein can have a significant effect upon its biological activity. That is, the oligosacchafides can affect the protein's antigenicity, stability, solubility and tertiary structure. The carbohydrate side-chains also can influence the protein's half-life and target it to receptors on the appropriate cells. The carbohydrate residues can affect both inter- and intra-cellular recognition. The sugar groups thus can control the relative effectiveness of a therapeutic protein when administrated to a patient. These and other such functions of the carbohydrate moiety of glycoproteins are discussed, for example, by Delente, Trends in Biotech. 3(9):218 (1985); van Brunt, Bio/Technology 4:835-839 (1986); and Taunton-Rigby, Biotech USA 1988, Proc. Conf. San Francisco, Nov. 14-16, pp. 168-176 (1988); and Varki, Glycobiology 3(2) 97-130 (1993).
Methods have also been developed for determining the sequence of oligosaccharides such as that described by Kobata in The Carbohydrates of Glycoproteins, Biology of Carbohydrates, (Ginsburg and Robins, Eds.), John Wiley and Sons, Vol. 2, pp. 87-162, (1984); Snider, ibid., pp. 163-193, 1984. See also Harada et al, Anal. Biochem. 164 374-381 (1987). Most proteins are glycoproteins which contain either O-gycosidically linked and/or N-gycosidically linked saccharides. These saccharides may vary from a single monosaccharide to highly branched structures containing over 30 monosaccharide residues. The determination of a monosaccharide sequence in such an oligosaccharide involves determining the order and branching pattern of the monosaccharide residues, the orientation of each glycosidic linkage (.alpha. or .beta.) and the linkage between the various monosaccharides, i.e. 1.fwdarw.3, 1.fwdarw.4, etc.
Most of the available enzymatic techniques for sequencing oligosaccharides are sequential in nature, that is, a single reaction is performed and its products are analyzed, followed by a second reaction and a second analysis, performed either on the starting material or on the products of the first reaction. The sequential analyses methods usually rely on enzymatic analysis using exoglycosidases. These enzymes specifically cleave the non-reducing terminal monosaccharide of an oligosaccharide.
These sequential enzymatic techniques have the advantage of great flexibility and sensitivity. That is, each subsequent reaction can be selected on the basis of the previous results, and the products of one reaction can be used as the starting point for the next. However, there also are disadvantages in these techniques in that the process can be slow, being a sequential technique, and difficult to automate unless the procedure is predefined, thereby resulting in loss of its flexibility. Other methods of oligosaccharide structure elucidation include H.sup.1 -NMR, mass spectrometry, and sequential lectin chromatography.
A recent improvement in sequential saccharide analysis is found in European Patent application EPO 421972 (Rademacher), for a technique dubbed Reagent Array Analysis Method (RAAM). The central idea is that when an oligosaccharide is exposed to a specific reagent mix having an array of oligosaccharide-cleaving enzymes, normally all linkages but one will cleave. When a reagent mix lacking the specific linkage enzyme is contacted with the oligosaccharide, no cleaving occurs, indicating which linkage is present. This approach lends itself to automation to a significant degree. However, if the oligosacharide bond is cleaved by more than one enzyme, a non-specific signal results.
The transfer of carbohydrate-conjugates from an electrophoresis gel to a membrane-based solid phase has been described in U.S. Pat. Nos. 5,019,231, 5,087,337, and 5,094,731 as the first step of a blotting application. Charged conjugate derivatives of the released oligosaccharides were derived by Schiffs' base formation between amine-containing charged fluorescent molecules such as 1-amino-4-naphthalene sulfonic acid (ANSA), 1-amino-1,6-disulphonic acid (ANDA) or 8-aminonapthalene-1,3,6-trisulfonic acid (ANTS). These sulphonated aminonaphthalenes only differ in their degree of sulfonic acid substitution, which in turn determines their total amount of ionic charge. The '231 and '731 patents are directed to a method of separating and analyzing saccharides by reacting saccharides with ANSA ('231) or more broadly, charge-generating and fiuorescing moities ('731 ), separating the conjugates on a gel, electroblotting the conjugates onto a nylon membrane, and probing the membrane-bound saccharide conjugates. Binding to the membrane is apparently difficult, because the charged ANSA tag was deficient in holding the neutral oligo-saccharide, and a secondary polyisobutylene methylmethacrylate polymer matrix was used to overcoat the adsorbed conjugates to bring about the necessary retention.
The previously mentioned '337 patent is directed to a method for separating and detecting saccharides by first reacting saccharides with a tri-functional compound, electrophoresing them, electroblotting them onto a nylon membrane, and then activating the light-sensitive azido group of the conjugate, which allows covalent binding to the nylon membrane. The tri-functional compound is a modified ANSA having an azido group at the 5, 6, 7, or 8 positions. The conjugate is attached to the membrane through light-activation of an azido group on the ANSA molecule. However, here the molecule, not the membrane, is activated. Also, these molecules are very light sensitive and have short shelf-lives.
U.S. Pat. No. 5,205,917 (Klock) is directed to fluorophore-assisted carbohydrate electrophoresis (FACE) used in a method of medical diagnosis. ANTS-carbohydrate derivatives are electrophoresed and recorded via charge-coupled device detector (CCD), or electroblotted onto nylon or nitrocellulose membranes. ANTS-carbohydrate analyses of individuals with glycoconjugate metabolic diseases are compared against normal people. This method does not attempt or result in covalent immobilization, and is directed to identification of metabolic disorders by comparison of patterns of electrophoresed carbohydrates.
Patent Cooperation Publication Application No. WO 91/05265 (Jackson) is directed to the use of a polymer membrane to blot electrophoresed ANTS-carbohydrate conjugates which have been run on an electrophoresis gel. Immobilon-N.TM. (Millipore Corporation), a cationically-coated PVDF membrane, is used to electroblot PAGE-separated ANTS-labeled sugars. The adsorption mechanism is believed to be electrostatic.
The need for better covalent immobilization of macromolecules to blotting membranes is apparent. There remains a need for proven methods and matrices for flexible means of immobilizing carbohydrates for further analysis, including sequencing.